Many methods utilizing a hybridization reaction using a probe carrier typified by a DNA microarray have been proposed as methods for rapidly and precisely determining the base sequence of a nucleic acid or detecting a target nucleic acid in a nucleic acid sample. The DNA microarray is that in which a probe including a nucleotide sequence that is complementary to a target nucleic acid is immobilized on a solid-phase such as a bead or a glass plate at a high density. In general, the detection of a target nucleic acid using the DNA microarray includes the following steps.
In the first step, a target nucleic acid is amplified by an amplification method typified by PCR. Specifically, firstly, first and second primers are added to a nucleic acid sample solution, and the mixture is subjected to a thermal cycle. The first primer specifically binds to part of the target nucleic acid, and the second primer specifically binds to part of a nucleic acid that is complementary to the target nucleic acid. The binding of the first and the second primers to a double-stranded nucleic acid containing the target nucleic acid causes amplification of the double-stranded nucleic acid containing the target nucleic acid by an extension reaction. After sufficient amplification of the double-stranded nucleic acid containing the target nucleic acid, a third primer is added to the nucleic acid sample solution, and the resulting mixture is subjected to a thermal cycle. The third primer is labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like and specifically binds to part of a nucleic acid that is complementary to the target nucleic acid. The binding of the third primer to the nucleic acid that is complementary to the target nucleic acid causes amplification of the target nucleic acid labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like by an extension reaction. That is, when the nucleic acid sample solution contains the target nucleic acid, the labeled target nucleic acid is generated. However, when the nucleic acid sample solution does not contain the target nucleic acid, no labeled target nucleic acid is generated.
In the second step, this nucleic acid sample solution is brought into contact with a DNA microarray for a hybridization reaction with a probe of the DNA microarray. The probe and the target nucleic acid form a hybrid when the nucleic acid sample solution contains a target nucleic acid that is complementary to the probe.
In the third step, the target nucleic acid is detected. Whether or not the probe and the target nucleic acid have formed a hybrid can be detected by means of the labeling substance of the labeled target nucleic acid. Thus, the presence or absence of a specific base sequence can be confirmed.
The DNA microarray utilizing a hybridization reaction is expected to be applied to medical diagnosis for identifying pathogenic microorganisms and to gene diagnosis for examining a patient for genetic constitution. However, in most cases, each step of amplification, hybridization, and detection of a nucleic acid is conducted by using its respective apparatuses. Consequently, the overall operation is complicated, and diagnosis hence takes a long time. In particular, in a case that a hybridization reaction is conducted on a slide glass, the probe-immobilizing surface is exposed. Accordingly, the probe may be lost or contaminated by touching the slide glass with a finger or the like. Therefore, careful handling is required. For the purpose of eliminating these problems, some biochemical reaction cassette structures in which a reaction chamber is provided with a DNA microarray so that a hybridization reaction and also a subsequent detection step can be performed in the reaction chamber have been proposed.
Japanese Patent Application Laid-Open No. 2003-302399 discloses a chamber structure for preventing air bubbles from remaining in the initial stage of filling with a liquid. Furthermore, Japanese Patent Application Laid-Open No. 2002-243748 discloses a structure for forming a uniform spread and a uniform flow of a liquid.
The reaction chambers of these biochemical reaction cassettes are usually low in height and have a flatly extended space, and the capacity thereof is small. Since the capacity of the reaction chamber is small, the amount of a liquid such as a nucleic acid sample solution used may be small. Since the height of the reaction chamber is low, a laminar flow is generated in the reaction chamber. In addition, the hybridization reaction of a target nucleic acid and a probe on a solid phase can be accelerated by agitating the nucleic acid sample solution in the reaction chamber. As the simplest way, the nucleic acid sample solution in the reaction chamber can be agitated by pushing and pulling the liquid at the injection port.
The reaction of a probe and a target nucleic acid should be uniformly conducted on a DNA microarray. Accordingly, it is necessary to decrease unevenness in the hybridization reaction by allowing fluid to uniformly flow in the reaction chamber.
The structure described in Japanese Patent Application Laid-Open No. 2003-302399 can allow a liquid to uniformly spread in a reaction chamber in the initial stage of being filled with the liquid, but the flow rate in the central portion of the reaction chamber becomes high in some cases when the liquid flows in the state that the reaction chamber is filled with the liquid.
Similarly, the structure described in Japanese Patent Application Laid-Open No. 2002-243748 causes a flow rate distribution in the reaction chamber in some cases when a liquid flows in the state that the reaction chamber is filled with the liquid.